Method for culturing adipose tissue-derived mesenchymal stem cells

Abstract

To provide methods for culturing adipose tissue-derived mesenchymal stem cells that can culture a large amount of adipose tissue-derived mesenchymal stem cells even with a small amount of adipose tissue.SOLUTION: A piece of adipose tissue surgically collected is adhered to a non-woven fabric treated with hydroxyapatite and cultured in a medium supplemented with autologous serum, the proliferated adipose tissue-derived mesenchymal stem cells are detached with trypsin, the detached adipose tissue-derived mesenchymal stem cells are washed and subsequently transferred to a flask to continue culturing, the adipose tissue attached to the trypsinized nonwoven fabric is washed, and the culturing is performed in a medium supplemented with autologous serum.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for culturing adipose tissue-derived mesenchymal stem cells. [Background technology]

[0002] Mesenchymal stem cells (MSCs) contained in bone marrow fluid and other fluids are known to have high proliferation capacity and to differentiate into various cells, such as bone, cartilage, muscle, fat, hepatocytes, nerve cells, cardiomyocytes, and vascular endothelial cells. Their usefulness as a cell source for regenerative medicine, particularly cell transplantation therapy, has been widely reported, and some of them have already been put into practical use, with further development for clinical applications underway.

[0003] Traditionally, MSCs have mainly been prepared by culturing bone marrow fluid. However, in recent years, adipose tissue has been attracting attention as a cell source for MSCs. This is because adipose tissue is rich in stem cells, is relatively easy to collect compared to bone marrow, and can be mass-produced when a large amount of tissue is obtained (Non-Patent Documents 1 to 5). Adipose tissue-derived MSCs generally proliferate faster than bone marrow-derived MSCs, making it relatively easy to secure the required number of cells (Non-Patent Document 6).

[0004] Adipose tissue is also harvested worldwide through liposuction for weight loss purposes, and if this is to be used as a source of cells for allogeneic transplantation, it is thought that there is a potentially large cell source. Thus, the effective use of adipose tissue is extremely important for the future development of regenerative medicine.

[0005] The preparation of therapeutic stem cells from adipose tissue is generally performed by treating surgically harvested adipose tissue with enzymes such as collagenase (animal derived), isolating the cells, and culturing them. Typically, 10-15 grams of adipose tissue material is used, and the number of therapeutic cells obtained by this method is 5-7 x 10 after one month of culture. 7 There are also reports that the degree of

[0006] When preparing stem cells such as MSCs from adipose tissue, fresh adipose tissue is used exclusively. For example, the isolation and culture of MSCs involves a series of steps, including washing the adipose tissue (removing attached blood, etc.), purifying it (removing excess tissue and blood vessels), treating it with enzymes such as proteases, filtering, centrifugation, and seeding the cells, before moving on to the cell culture process.

[0007] Therefore, if a large amount of fresh adipose tissue is obtained, it may not be possible to use the fresh adipose tissue for processing up to the cell culture step, and the obtained fresh tissue may not be fully utilized and may have to be discarded. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] 1.Lee KD, et al.: In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology. 2004;40:1153-61. [Non-patent document 2] 2. Hong SH, et al.: In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells. Biochem Biophys Res Commun. 2005;330:1153-61. [Non-patent document 3] 3. Teramoto K, et al.: Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells. J Hepatobiliary Pancreat Surg. 2005;12:196-202. [Non-patent document 4] 4. Lee OK, et al.: Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 2004;103:1669-75. [Non-Patent Document 5] 5. Yang SE, et al.: Mesenchymal stem / progenitor cells developed in cultures from UC blood. Cytotherapy 2004;6:476-86. [Non-patent document 6] 6. Nakayama T, et al.: Cell therapy using adipose-derived mesenchymal stromal cells: current status and perspectives. Japanese Journal of Transfusion and Cell Therapy. 2013;59:450-56. [Non-Patent Document 7] 7. Mendell JR, et al.: Gene therapy for muscular dystrophy: lessons learned and path forward. Neurosci Lett. 2012;527:90-9. [Non-patent document 8] 8. Fraser JK, et al.: Fat tissue: an underappreciated source of stem cells for biotechnology. 2006;24:150-4. [Non-Patent Document 9] 9. Ronti T, et al: The endocrine function of adipose tissue: an update. Clin Endocrinol (Oxf). 2006;64:355-65. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of such problems, and aims to provide a method for culturing adipose tissue-derived mesenchymal stem cells that can culture large quantities of adipose tissue-derived mesenchymal stem cells even from a small amount of adipose tissue. [Means for solving the problem]

[0010] The method for culturing adipose tissue-derived mesenchymal stem cells according to the present invention comprises: (i) an isolation and culture step in which 0.1 to 1.0 g of surgically collected adipose tissue is chopped into adipose tissue fragments without enzyme treatment, the adipose tissue fragments are attached to a nonwoven fabric treated with hydroxyapatite, and cultured in a medium containing no FBS and containing 1 to 2% by weight of autologous serum; (ii) a first enzyme treatment step in which, after the isolation and culture step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached using trypsin; (iii) a first expansion culture step in which, after the first enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask and continued to be cultured in a serum-free medium; (iv) a re-culture step in which the adipose tissue adhering to the nonwoven fabric treated with trypsin in the first enzyme treatment step is washed and cultured in a medium containing no FBS and containing 1 to 2% by weight of autologous serum; (v) a second enzyme treatment step in which the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached using trypsin after the re-culture step; (vi) a second expansion culture step in which, after the second enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask and continued to be cultured in a serum-free medium; (vii) A method for culturing adipose tissue-derived mesenchymal stem cells, characterized in that adipose tissue-derived mesenchymal stem cells are obtained in the first expansion culture step and the second expansion culture step. [Effects of the Invention]

[0011] According to the present invention, it is possible to culture a large amount of adipose tissue-derived mesenchymal stem cells. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 2 is a photograph showing the state of culturing adipose tissue-derived mesenchymal stem cells in an autologous serum-containing medium in the isolation and culture step (step (i)). [Figure 2] FIG. 1 is a photograph showing the state in which the proliferated cells have come to occupy 10 to 20% of the nonwoven fabric substrate in the first enzyme treatment step (step (ii)). [Figure 3] FIG. 10 is a photograph showing the state in which adipose tissue-derived mesenchymal stem cells detached with trypsin are transferred to a plastic flask and the culture of the adipose tissue-derived mesenchymal stem cells is continued in the first expansion culture step (step (iii)). [Figure 4] FIG. 10 is a photograph showing a state in which adipose tissue-derived mesenchymal stem cells have grown in a monolayer attached to the bottom surface of a plastic flask in the first expansion culture step (step (iii)). [Figure 5] FIG. 1 is a photograph showing the state in which the adipose tissue remaining on the nonwoven fabric that has been treated with trypsin in the first enzyme treatment step (step (ii)) is washed with physiological saline. [Figure 6] FIG. 1 shows the results of analyzing the expression pattern of cell membrane surface antigens using cells at passage 4 when cells grown on nonwoven fabric were detached for the first time by trypsin treatment. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

[0014] The method for culturing adipose tissue-derived mesenchymal stem cells according to the present invention comprises the following steps.

[0015] Step (i) Separation and culture step Step (ii) First enzyme treatment step Step (iii) First expansion culture step Step (iv) Re-culture step Step (v) Second enzyme treatment step Step (vi) Second expansion culture step

[0016] 1. Process (i) Separation and culture process In the isolation and culture process, surgically collected adipose tissue fragments are attached to a nonwoven fabric treated with hydroxyapatite and cultured in a medium supplemented with autologous serum.

[0017] Adipose tissue includes any adipose tissue and may be from any organism that has adipose tissue, preferably the adipose tissue is mammalian, and most preferably the adipose tissue is human.

[0018] Adipose tissue fragments are surgically harvested from adipose tissue without using enzymes such as collagenase. A small amount of adipose tissue is sufficient, and although not particularly limited, it is, for example, 0.02 to 1.0 g, preferably 0.1 to 1.0 g. The harvested adipose tissue is cut into approximately four strips to produce adipose tissue fragments, which are then placed on a substrate such as a cell strainer, and blood and preservative solution adhering to the adipose tissue fragments are washed away with PBS.

[0019] Unlike regularly woven materials such as mesh, nonwoven fabrics have fiber branches that are irregularly connected, providing just the right amount of space, thickness, and elasticity. Therefore, when cells adhere to each other in areas where they are not in contact with the scaffold (nonwoven fabric), the nonwoven fabric has the function of maintaining an appropriate distance between the cells, promoting adhesion between the cells.

[0020] Examples of raw materials for nonwoven fabrics include natural fibers (cotton, linen, wool, etc.), regenerated fibers (rayon, cupra), semi-synthetic fibers (acetate, promix), synthetic fibers (nylon, polyester, acrylic, vinylon, polyvinyl chloride, vinylidene, polyolefin, polyurethane, polychlar, fluorocarbon, novoloid, etc.), inorganic fibers (glass fiber, carbon fiber, alumina fiber, silicon carbide fiber, slag fiber, metal fiber, etc.), etc.

[0021] The fiber density of the nonwoven fabric is not particularly limited, but is, for example, 0.1 to 0.3 g / 100 cm 2 is.

[0022] The method for treating the nonwoven fabric with hydroxyapatite is not particularly limited, but examples thereof include a method of applying hydroxyapatite to the nonwoven fabric, and a method of bonding the nonwoven fabric with a binder (adhesive).

[0023] The hydroxyapatite-treated nonwoven fabric (also referred to as the nonwoven fabric substrate) is hydrophilized by, for example, immersing it in 80% alcohol for several minutes, and then washing it with PBS, after which the adipose tissue fragments are attached to the nonwoven fabric substrate.

[0024] The cells are cultured in a medium supplemented with autologous serum. Specifically, the culture is performed in a commercially available medium supplemented with supplements, etc., with autologous serum added without the use of FBS. The autologous serum is at a low concentration, not particularly limited, but for example, 1-2% by weight. After culturing for 1-2 weeks in a medium supplemented with autologous serum, the cells begin to proliferate. However, it is difficult to induce proliferation for cell isolation using a commercially available medium supplemented with supplements without using autologous serum.

[0025] 2. Step (ii) First Enzyme Treatment Step In the first enzyme treatment step, after the isolation and culture step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached with trypsin (first detachment).

[0026] That is, when the cells occupy, for example, 10 to 20% of the nonwoven fabric substrate, the cells are detached from the nonwoven fabric substrate by trypsin treatment using a trypsin solution. In the present invention, trypsin refers to a protease secreted by the pancreas or a degrading enzyme with properties equivalent to those of the protease. Examples of commercially available trypsin include Trypsin™, TrypLE™ SELECT, and TrypLE™ EXPRES (all manufactured by Thermo Fisher Scientific).

[0027] The trypsin solution used in the trypsin treatment may contain not only trypsin but also an enzyme having a proteolytic activity similar to trypsin, as well as a chelating agent, etc. The trypsin content in the trypsin solution is not particularly limited, but is preferably 0.01 to 2.5 wt%, more preferably 0.1 to 1 wt%, particularly preferably 0.2 to 0.5 wt%, and most preferably 0.25 to 0.35 wt%.

[0028] 3. Step (iii) First expansion culture step In the first expansion culture step, after the first enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask for continued culture.

[0029] Specifically, cells are detached by trypsinization, washed, and then subcultured in flasks. After subculture in flasks, they can be cultured in a commercially available serum-free medium supplemented with supplements.

[0030] 4. Step (iv) Re-culture step That is, in the cultivation step, the adipose tissue adhering to the nonwoven fabric that has been treated with trypsin in the first enzyme treatment step is washed away, and the cells are cultured in a medium containing autologous serum.

[0031] The culture in step (iv) is carried out in a medium supplemented with autologous serum, as in step (i). The concentration of the autologous serum is, for example, 1 to 2% by weight, as in step (i). After culturing in a medium supplemented with autologous serum for 1 to 2 weeks, the cells begin to proliferate.

[0032] 5. Step (v) Second Enzyme Treatment Step That is, after the re-culturing step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached with trypsin (second detachment).

[0033] That is, when the cells occupy, for example, 8 to 20% of the nonwoven fabric substrate, the cells are detached from the nonwoven fabric substrate by trypsin treatment using a trypsin solution, as in step (ii). The trypsin content in the trypsin solution is not particularly limited, but is preferably 0.01 to 2.5 wt%, more preferably 0.1 to 1 wt%, particularly preferably 0.2 to 0.5 wt%, and most preferably 0.25 to 0.35 wt%, as in step (ii).

[0034] 6. Step (vi) Second expansion culture step That is, after the second enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask for continued culture. After subculture in a flask, as in step (iii), they can be cultured solely in a commercially available serum-free medium supplemented with supplements.

[0035] 7. Stem Cell Obtainment In the present invention, adipose tissue-derived mesenchymal stem cells are obtained in this manner through the first expansion culture step and the second expansion culture step. In the present invention, the process can be terminated at the aforementioned step (vi), or it is also possible to further wash the adipose tissue remaining on the trypsin-treated nonwoven fabric in the aforementioned step (v) with physiological saline, culture the adipose tissue-derived mesenchymal stem cells in a serum-free medium supplemented with 1 to 2% by weight of autologous serum, and then detach the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric with trypsin (third detachment) (trypsin treatment), wash the detached adipose tissue-derived mesenchymal stem cells, and then transfer them to a flask to continue culturing the adipose tissue-derived mesenchymal stem cells. It is also possible to further repeat these steps multiple times.

[0036] The expression of surface antigens on the adipose tissue-derived mesenchymal stem cells obtained by the first detachment in step (ii) and the adipose tissue-derived mesenchymal stem cells obtained by the second detachment in step (v) is the same as that on existing adipose tissue-derived mesenchymal stem cells. Furthermore, the expression of surface antigens on adipose tissue-derived mesenchymal stem cells obtained by the third, fourth, or subsequent detachments is also the same as that on existing adipose tissue-derived mesenchymal stem cells. Thus, according to the present invention, large amounts of adipose tissue-derived mesenchymal stem cells with the same performance as conventional ones can be obtained from a small amount of adipose tissue. [Example]

[0037] 1. Cultivation method of adipose tissue-derived mesenchymal stem cells 1-1. Isolation and culture process (step (i)) Adipose tissue fragments (0.5 g) were surgically harvested without the use of enzymes. A commercially available hydroxyapatite-loaded nonwoven fabric (a hydroxyapatite-coated polyethylene-polypropylene (PE-PP) core-sheath nonwoven fabric) was used. This nonwoven fabric substrate was immersed in 80% alcohol for several minutes and washed three times with injectable saline. The harvested adipose tissue fragments were then attached to the hydroxyapatite-treated nonwoven fabric. Adipose tissue-derived mesenchymal stem cells were then cultured in a medium (Figure 1). This medium was a commercially available medium (MSC NutriStem® XF Medium: Biological Industries, Israel) supplemented with a supplement (MSC NutriStem® XF Supplement Mix: Biological Industries, Israel) and 1% by weight of autologous serum. Culture conditions were 37°C and 5% CO2 in a humidified atmosphere.

[0038] 1-2. First enzyme treatment step (step (ii)) Next, after culturing for one week, the proliferated cells occupied 10-20% of the nonwoven substrate (Figure 2). After that, the adipose tissue-derived mesenchymal stem cells that had attached and proliferated on the nonwoven substrate were detached with trypsin (first detachment) (trypsin treatment). The trypsin used was a recombinant trypsin / EDTA solution.

[0039] 1-3. First expansion culture step (step (iii)) Next, the adipose tissue-derived mesenchymal stem cells were detached with trypsin, washed, and then transferred to a plastic flask (T75 flask) for continued culturing (Figure 3). After subculture in the plastic flask, the cells were cultured solely in serum-free MSC NutriStem® XF Medium supplemented with supplements. The adipose tissue-derived mesenchymal stem cells adhered to the bottom of the plastic flask and proliferated in a monolayer (Figure 4).

[0040] 1-4. Re-cultivation process (step (iv)) Next, the adipose tissue remaining on the nonwoven fabric that had been trypsinized in step (ii) above was washed with saline (Figure 5), and adipose tissue-derived mesenchymal stem cells were cultured in serum-free medium supplemented with 1% by weight of autologous serum.

[0041] 1-5. Second enzyme treatment step (step (v)) Next, after the re-culturing process, the adipose tissue-derived mesenchymal stem cells that had adhered to and proliferated on the nonwoven fabric were detached (second detachment) using trypsin (trypsin treatment). The trypsin used was a recombinant trypsin / EDTA solution.

[0042] 1-6. Second expansion culture step (step (vi)) Next, after the second enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells were washed and then transferred to a plastic flask, where the adipose tissue-derived mesenchymal stem cells were continued to be cultured.

[0043] In this example, the above steps were repeated multiple times to efficiently culture adipose tissue-derived mesenchymal stem cells.

[0044] That is, in this example, it is possible to end the process at the aforementioned step (vi), or it is also possible to further wash the adipose tissue remaining on the nonwoven fabric that was trypsinized in the aforementioned step (v) with saline, and then culture the adipose tissue-derived mesenchymal stem cells in a serum-free medium supplemented with 1% autologous serum, and then detach the adipose tissue-derived mesenchymal stem cells that have attached and proliferated on the nonwoven fabric with trypsin (third detachment) (trypsin treatment), and after washing the detached adipose tissue-derived mesenchymal stem cells, transfer them to a plastic flask to continue culturing the adipose tissue-derived mesenchymal stem cells, and it is also possible to repeat these steps multiple times.

[0045] In this example, the cells isolated from the adipose tissue treated with trypsin after the second treatment could be cultured in the same way as the first treatment. This procedure could be continued for about 4 months at intervals of at least 1-2 weeks. After one month of culture, 1 × 10 cells were isolated from a batch of cells treated with trypsin. 8It was possible to culture the above adipose tissue-derived mesenchymal stem cells.

[0046] 2. Characteristics of adipose tissue-derived mesenchymal stem cells obtained in this example Surface antigen expression analysis was performed on adipose tissue-derived mesenchymal stem cells obtained by the first detachment in the aforementioned step (ii), adipose tissue-derived mesenchymal stem cells obtained by the second detachment in the aforementioned step (v), and adipose tissue-derived mesenchymal stem cells obtained by the third detachment (Table 1).

[0047] [Table 1]

[0048] In Table 1, for example, 1-3p refers to the third passage when cells grown on nonwoven fabric were first detached by trypsinization. Analysis of cell surface antigen expression revealed that CD34, CD45, HLA-DR, CD11c, CD80, and CD56 were negative, and CD44, CD73, CD90, and CD105 were positive. This cell surface antigen expression pattern was similar in the sixth passage of cells detached the first time, the second passage of cells detached the second time, and the fifth passage of cells detached the third time.

[0049] Next, adipose tissue-derived mesenchymal stem cells were isolated and cultured from the adipose tissue of three volunteers, and surface antigen expression analysis was performed (Table 2).

[0050] [Table 2]

[0051] In Table 2, for example, 1-4p refers to the fourth passage of cells grown on nonwoven fabric, which were first detached by trypsinization. Analysis of the expression pattern of cell membrane surface antigens using cells at passage 4 after the first separation revealed positive results for CD44, CD73, CD90, and CD105, but negative results for CD34, CD45, and HLA-DR (Figure 6). In Figure 6, lightly colored histograms indicate negative background, while darkly colored histograms indicate positive antigen binding. This is consistent with many published reports that indicate the surface antigen expression pattern of adipose tissue-derived stem cells. Thus, analysis of the cell surface antigen expression pattern of the cells obtained in this example was consistent with data reported in the literature, confirming that they possess the properties of adipose tissue-derived stem cells. [Industrial Applicability]

[0052] It can be used to culture adipose tissue-derived mesenchymal stem cells.

Claims

1. (i) an isolation and culture step in which 0.1 to 1.0 g of surgically collected adipose tissue is chopped into adipose tissue fragments without enzyme treatment, the adipose tissue fragments are attached to a nonwoven fabric treated with hydroxyapatite, and cultured in a medium containing no FBS and containing 1 to 2% by weight of autologous serum; (ii) a first enzyme treatment step in which, after the isolation and culture step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached using trypsin; (iii) a first expansion culture step in which, after the first enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask and continued to be cultured in a serum-free medium; (iv) a re-culture step in which the adipose tissue adhering to the nonwoven fabric treated with trypsin in the first enzyme treatment step is washed and cultured in a medium containing no FBS and containing 1 to 2% by weight of autologous serum; (v) a second enzyme treatment step in which, after the re-culture step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached using trypsin; (vi) a second expansion culture step in which, after the second enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask and continued to be cultured in a serum-free medium; (vii) Adipose tissue-derived mesenchymal stem cells are obtained in the first expansion culture step and the second expansion culture step. A method for culturing adipose tissue-derived mesenchymal stem cells.

2. (i) An isolation and culture process in which surgically collected adipose tissue fragments are attached to a nonwoven fabric treated with hydroxyapatite and cultured in a medium containing autologous serum; (ii) a first enzyme treatment step in which, after the isolation and culture step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached using trypsin; (iii) a first expansion culture step in which, after the first enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask for continued culture; (iv) a re-culture step in which the adipose tissue adhering to the nonwoven fabric treated with trypsin in the first enzyme treatment step is washed and cultured in a medium containing autologous serum; (v) a second enzyme treatment step in which, after the re-culture step, the adipose tissue-derived mesenchymal stem cells that have adhered to and proliferated on the nonwoven fabric are detached using trypsin; (vi) a second expansion culture step in which, after the second enzyme treatment step, the detached adipose tissue-derived mesenchymal stem cells are washed and then transferred to a flask for continued culture; (vii) Adipose tissue-derived mesenchymal stem cells are obtained in the first expansion culture step and the second expansion culture step. A method for culturing adipose tissue-derived mesenchymal stem cells.

3. 3. The method for culturing adipose tissue-derived mesenchymal stem cells according to claim 2, wherein the first expansion culture step is carried out in a serum-free medium.

4. 4. The method for culturing adipose tissue-derived mesenchymal stem cells according to claim 2 or 3, wherein the second expansion culture step is carried out in a serum-free medium.

5. A method for culturing adipose tissue-derived mesenchymal stem cells described in any one of claims 2 to 4, characterized in that in the isolation and culture process, 0.1 to 1.0 g of surgically collected adipose tissue is chopped into adipose tissue fragments without enzymatic treatment.

6. A method for culturing adipose tissue-derived mesenchymal stem cells according to any one of claims 2 to 5, characterized in that in the isolation and culture process, the culture is carried out in a medium that does not contain FBS and to which 1 to 2% by weight of autologous serum is added.

7. 7. A method for culturing adipose tissue-derived mesenchymal stem cells according to any one of claims 2 to 6, characterized in that the re-culturing step is carried out in a medium that does not contain FBS and to which 1 to 2% by weight of autologous serum is added.

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